Aims. This study intended to investigate the effect of vericiguat (VIT) on titanium rod osseointegration in aged rats with iron overload, and also explore the role of VIT in osteoblast and osteoclast differentiation. Methods. In this study, 60 rats were included in a titanium rod implantation model and underwent subsequent guanylate cyclase treatment. Imaging, histology, and biomechanics were used to evaluate the osseointegration of rats in each group. First, the impact of VIT on bone integration in aged rats with iron overload was investigated. Subsequently, VIT was employed to modulate the differentiation of MC3T3-E1 cells and RAW264.7 cells under conditions of iron overload. Results. Utilizing an OVX rat model, we observed significant alterations in bone mass and osseointegration due to VIT administration in aged rats with iron overload. The observed effects were concomitant with reductions in bone metabolism, oxidative stress, and inflammation. To elucidate whether these effects are associated with osteoclast and osteoblast activity, we conducted in vitro experiments using MC3T3-E1 cells and RAW264.7 cells. Our findings indicate that iron accumulation suppressed the activity of MC3T3-E1 while enhancing RAW264.7 function. Furthermore, iron overload significantly decreased oxidative stress levels; however, these detrimental effects can be mitigated by VIT treatment. Conclusion. Collectively, our data provide compelling evidence that VIT has the potential to reverse the deleterious consequences of iron overload on osseointegration and bone mass during ageing. Cite this article: Bone Joint Res 2024;13(9):427–440

Aseptic inflammation is the main factor causing aseptic loosening of artificial joints. Studies have shown that inflammatory cells can activate STING (stimulator of interferon genes, STING) after being stressed. This study aims to explore the specific mechanism of STING in aseptic loosening of artificial joints, and provide new strategies for disease prevention. Titanium particles with a diameter of 1.2-10 μm were prepared to stimulate macrophages (RAW 264.7) to simulate the periprosthetic microenvironment. A lentiviral vector targeting the STING gene was designed and transfected into macrophages to construct a cell line targeting STING knockdown. The expression and secretion levels of TNF-α were detected by qPCR and ELISA, the activation levels of inflammatory pathways (NF-κB, IRF3, etc.) were detected by western blot, and the nucleus translocation of P65 and IRF3 was observed by cellular immunofluorescence. After titanium particles stimulated macrophages, qPCR and ELISA showed that the transcription and secretion levels of TNF-α were significantly increased. Western blot showed that titanium particle stimulation could increase the phosphorylation levels of NF-κB and IRF3 pathways. While knockdown of STING can significantly reduce titanium particle-induced TNF production, attenuate the activation levels of NF-κB and IRF3 pathways as well as the nucleus translocation of P65 and IRF3. Conclusions: STING positively regulates the level of inflammation in macrophages induced by titanium particles, and targeted inhibition of STING can reduce inflammation, which may delay the progression of aseptic loosening of artificial joints

Introduction and Objective. Intervertebral disc (IVD) degeneration is one of the major contributors to low back pain, the leading cause of disability worldwide. This multifactorial pathological process involves the degradation of the extracellular matrix, inflammation, and cell loss due to apoptosis and senescence. While the deterioration of the extracellular matrix and cell loss lead to structural collapse of the IVD, increased levels of inflammation result in innervation and the development of pain. Amongst the known regulators of inflammation, toll-like receptors (TLRs) and more specifically TLR-2 have been shown to be specifically relevant in IVD degeneration. As strong post-transcriptional regulators, microRNAs (miRNAs) and their dysregulation has been connected to multiple pathologies, including degenerative diseases such as osteoarthritis and IVD degeneration. However, the role of miRNAs in TLR signalling in the IVD is still poorly understood and was hence investigated in this study. Materials and Methods. Human Nucleus pulposus (hNP) and Annulus fibrosus (hAF) cells (n=5) were treated with the TLR-2/6 specific agonist PAM2CSK4 (100 ng/mL for 6 hours) in order to activate the TLR2 signalling pathway. After the activation both miRNA and mRNA were isolated, followed by next-generation sequencing and qPCR analysis of proinflammatory cytokines respectively. Furthermore, cell supernatants were used to analyze the secretion of proinflammatory cytokines with enzyme-linked immunosorbent assay. TLR-2 knockdown (siRNA) cells were used as a control. Statistical analysis was conducted by performing Kolmogorov-Smirnov test and a two-tailed Student's t-test using GraphPad Prism version 9.0.2 for Windows (GraphPad Software, La Jolla California USA). Results. TLR-2 activation resulted in the induction of an inflammatory cell response, with a significant increase in gene expression of interleukin (IL)-6 (525 ± 180 fold change, p < 0.05) and IL-8 (7513 ± 1907 fold change, p < 0.05) and protein secretion of IL-6 (30.5 ± 8.1 pg/mL) and IL-8 (28.9 ± 5.4 pg/mL). TLR-2 activation was furthermore associated with changes in the miRNA profile of hNP and hAF cells. Specifically, we identified 10 differentially expressed miRNAs in response to TLR-2 activation, amongst which were miR-335–3p (1.45 log2 FC, p < 0.05), miR-125b-1–3p (0.55 log2 FC, p < 0.05), and miR-181a-3p (−1.05 log2 FC, p < 0.05). Conclusions. The identified miRNAs are known to be associated with osteoarthritis (miR-335-3p), inflammation and IVD degeneration (mir-125-1-3p and miR-181a-3p), but the link to TLR signalling has not been previously reported. Experiments to validate the identified miRNAs and elucidate their functional role are undergoing. The identification of these miRNAs provides an opportunity to further investigate miRNAs in the context of TLR activation and inflammation and to enhance our understanding of underlying molecular mechanisms behind disc degeneration, inflammation, and TLR dysregulation

As peri-prosthetic aseptic loosening is one of the main causes of implant failure, inhibiting wear particles induced macrophages inflammation is considered as a promising therapy for AL to expand the lifespan of implant. Here, we aim at exploring the role of p110δ, a member of class IA PI3K family, and Krüppel-like factor 4 (KLF4) in titanium particles (TiPs) induced macrophages-inflammation and osteolysis. Firstly, IC87114, the inhibitor of p110δ and siRNA targeting p110δ were applied and experiments including ELISA and immunofluorescence assay were conducted to explore the role of p110δ. Sequentially, KLF4 was predicted as the transcription factor of p110δ and the relation was confirmed by dual luciferase reporter assay. Next, assays including RT-PCR, western blotting and flow cytometry were performed to ensure the specific role of KLF4. Finally, TiPs-induced mice cranial osteolysis model was established, and micro-CT scanning and immunohistochemistry assay were performed to reveal the role of p110δ and KLF4 in vivo. Here, we found that p110δ was upregulated in TiPs-stimulated macrophages. The inhibition of p110δ or knockdown of p110δ could significantly dampen the TiPs-induced secretion of TNFα and IL-6. Further mechanistic studies confirmed that p110δ was responsible for TNFα and IL-6 trafficking out of Golgi complex without affecting their expression in TiPs-treated macrophages. Additionally, we explored the upstream regulators and confirmed that Krüppel-like factor 4 (KLF4) was the transcription repressor of p110δ. Apart from that, KLF4, targeted by miR-92a, could also attenuate TiPs-induced inflammation by mediating NF-κB pathway and M1/M2 polarization. By the establishment of TiPs-induced mice cranial osteolysis model, we found that KLF4 knockdown exacerbated TiPs-induced osteolysis which was strikingly ameliorated by knockdown of p110δ. In summary, our study suggests the key role of miR-92a/KLF4/p110δ signal in TiPs-induced macrophages inflammation and osteolysis

Tendinopathy is the most frequent musculoskeletal disease that requires medical attention. Mechanical overload has been considered as a key driver of its pathology. However, the underline mechanism on how overload induces tendinopathy and inflammation is unclear. Extracellular mitochondria (EM) are newly identified as cell-to-cell communicators. The aim of this study is to elucidate the role of mitochondria in overload-induced inflammation. We performed three-dimensional uniaxial stretching to mouse tendon organoid in bioreactors. Cyclic strain of uniaxial loadings included underload, normal load, and overload, according to previous work. We then harvested microvesicles including EM, from the bioreactor by differential centrifugation and evaluated their characteristics by flow cytometry and super-resolution confocal microscopy. Raw 264.7 mouse macrophage cell line was used for chemotaxis assay in a Boyden Chamber System with Magnetic-Activated Cell Sorting Technology. EM induced cytokines secretion by macrophages was analyzed by a bead-based multiplex assay panel. N-Acetyl-L-cysteine (NAC) was used as the antioxidant to tendon organoid to regulate mitochondrial fitness. We showed mechanical load induced tendon organoid to release microvesicles including mitochondria. The size of microvesicles is mainly in the range from 220nm to 880nm. More than 75% of microvesicles could be stained by PKH26, confirming they were with lipophilic membrane. Super-resolution confocal microscopy identified two forms of mitochondria, including mitochondria encapsulated in vesicles and free mitochondria. Overload led to the degeneration of the organoid and induced microvesicles release containing most EM. Chemotaxis assay showed that EM from overloaded tendon organoid induced macrophages chemotaxis. In addition, microvesicles extracted from overloaded tendon organoid induced the production of proinflammatory cytokines including IL-6, KC (Keratinocyte-Derived Chemokine) and IL-18. NAC treatment to tendon cells could attenuate overload-induced macrophage chemotaxis. Overload induces EM releasing from tendon cells, which leads to chemotaxis of macrophages toward tendon, resulting in induction of inflammation

MicroRNA (miR) delivery to regulate chronic inflammation hold extraordinary promise, with new therapeutic possibilities emanating from their ability to fine-tune multiple target gene regulation pathways which is an important factor in controlling aberrant inflammatory reactions in complex multifactorial disease. However, several hurdles have prevented advancements in miR-based therapies. These include off-target effects of miRs, limited trafficking, and inefficient delivery. We propose a magnetically guided nanocarrier to transport therapeutically relevant miRs to assist self- resolving inflammation processes at injury sites and reduce the impact of chronic inflammation- related diseases such as tendinopathies. The high prevalence, significant socio-economic burden and increasing recognition of dysregulated immune mediated pathways in tendon disease provide a compelling rationale for exploring inflammation-targeting strategies as novel treatments in this condition. By combining cationic polymers, miR species (e.g., miR 29a, miR155 antagonist), and magnetic nanoparticles in the form of magnetoplexes with highly efficient magnetofection procedures, we developed inexpensive, easy-to-fabricate, and biocompatible systems with competent miR-binding and fast cellular uptake into different types of human cells, namely macrophages and tendon-derived cells. The system was shown to be cell-compatible and to successfully modulate the expression and production of inflammatory markers in tendon cells, with evidence of functional pro-healing changes in immune cell phenotypes. Hence, magnetoplexes represent a simple, safe, and non-viral nanoplatform that enables contactless miR delivery and high- precision control to reprogram cell profiles toward improved pro-regenerative environments. Acknowledgements: ERC CoG MagTendon No.772817; FCT Doctoral Grant SFRD/BD/144816/2019, and TERM. RES Hub (Norte-01-0145-FEDER-022190)

Osteoarthritis (OA) is a degenerative joint disease affecting millions worldwide. Early detection of OA and monitoring its progression is essential for effective treatment and for preventing irreversible damage. Although sensors have emerged as a promising tool for monitoring analytes in patients, their application for monitoring the state of pathology is currently restricted to specific fields (such as diabetes). In this study, we present the development of an optical sensor system for real-time monitoring of inflammation based on the measurement of nitric oxide (NO), a molecule highly produced in tissues during inflammation. Single-walled carbon nanotubes (SWCNT) were functionalized with a single-stranded DNA (ssDNA) wrapping designed using an artificial intelligence approach and tested using S-nitroso-N-acetyl penicillamine (SNAP) as a standard released-NO marker. An optical SWIR reader with LED excitation at 650 nm, 730 nm and detecting emission above 1000 nm was developed to read the fluorescence signal from the SWCNTs. Finally, the SWCNT was embedded in GelMa to prove the feasibility of monitoring the release of NO in bovine chondrocyte and osteochondral inflamed cultures (1–10 ng/ml IL1β) monitored over 48 hours. The stability of the inflammation model and NO release was indirectly validated using the Griess and DAF-FM methods. A microfabricated sensor tag was developed to explore the possibility of using ssDNA-SWCNT in an ex vivo anatomic set-up for surgical feasibility, the limit of detection, and the stability under dynamic flexion. The SWCNT sensor was sensitive to NO in both in silico and in vitro conditions during the inflammatory response from chondrocyte and osteochondral plug cultures. The fluorescence signal decreased in the inflamed group compared to control, indicating increased NO concentration. The micro-tag was suitable and stable in joints showing a readable signal at a depth of up to 6 mm under the skin. The ssDNA-SWCNT technology showed the possibility of monitoring inflammation continuously in an in vitro set-up and good stability inside the joint. However, further studies in vivo are needed to prove the possibility of monitoring disease progression and treatment efficacy in vivo. Acknowledgments: The project was co-financed by Innosuisse (grant nr. 56034.1 IP-LS)

In osteoarthritis, chondrocytes acquire a hypertrophic phenotype that contributes to matrix degradation. Inflammation is proposed as trigger for the shift to a hypertrophic phenotype. Using in vitro culture of human chondrocytes and cartilage explants we could not find evidence for a role of inflammatory signalling activation. We found, however, that tissue repair macrophages may contribute to the onset of hypertrophy (doi: 10.1177/19476035211021907) Intra-articularly injected triamcinolone acetonide to inhibit inflammation in a murine model of collagenase-induced osteoarthritis, increased synovial macrophage numbers and osteophytosis, confirming the role of macrophages in chondrocyte hypertrophy occurring in osteophyte formation (doi: 10.1111/bph.15780). In search of targets to inhibit chondrocyte hypertrophy, we combined existing microarray data of different cartilage layers of murine growth plate and murine articular cartilage after induction of collagenase-induced osteoarthritis. We identified common differentially expressed genes and selected those known to be associated to inflammation. This revealed EPHA2, a tyrosine kinase receptor, as a new target. Using in silico, in vitro and in vivo models we demonstrated that inhibition of EPHA2 might be a promising treatment for osteoarthritis. Recently, single cell RNA-seq. has revealed detailed information about different populations of chondrocytes in articular cartilage during osteoarthritis. We re-analysed a published scRNA-seq data set of healthy and osteoarthritic cartilage to obtain the differentially expressed genes in the population of hypertrophic chondrocytes compared to the other chondrocytes, applied pathway analyses and then used drug databases to search for upstream inhibitors of these pathways. This drug repurposing approach led to the selection of 6 drugs that were screened and tested using several in vitro models with human chondrocytes and cartilage explants. In this lecture I will present this sequence of studies to highlight different approaches and models that can be used in the quest for a disease modifying drug for osteoarthritis

Vascular inflammation and activation of myofibroblasts are significant contributors to the progression of fibrosis, which can severely impair tissue function. In various tissues, including tendons, Transforming growth factor beta 1 (TGF-β1) has been identified as a critical driver of adhesion and scar formation. Nevertheless, the mechanisms that underlie fibrotic peritendinous adhesions are still not well comprehended, and human microphysiological systems to help identify effective therapies remain scarce. To address this issue, we developed a novel human Tendon-on-a-Chip (hToC), comprised of an endothelialized vascular compartment harboring circulating monocytes and separated by a 5 μm/100 nm dual-scale ultrathin porous membrane from a type I/III collagen hydrogel with primary tendon fibroblasts and tissue-resident macrophages, all under defined serum-free conditions. The hToC models the crosstalk of the various cells in the system leading to the induction of inflammatory and fibrotic pathways including the activation of mTOR signaling. Consistent with phenotypes observed in vivo in mouse models and clinical human samples, we observed myofibroblast differentiation and senescence, tissue contraction, excessive extracellular matrix deposition, and monocytes’ transmigration and macrophages’ secretion of inflammatory cytokines, which were dependent on the presence of the endothelial barrier. This model offers novel insights on the role of vasculature in the pathophysiology of adhesions, which were previously underappreciated. Moreover, in testing whether the hToC could be used to evaluate efficacy of therapeutics, we were able to capture donor-specific variability in the response to Rapamycin treatment, which reduced myofibroblast activation regardless. Thus, our findings demonstrate the value of the hToC as a human microphysiological system for investigating the pathophysiology of fibrotic conditions in the context of peritendinous injury and similar fibrotic conditions, providing an alternative to animal testing

Objectives. The aim of this study was to investigate the role of miR-126 in the development of osteoarthritis, as well as the potential molecular mechanisms involved, in order to provide a theoretical basis for osteoarthritis treatment and a novel perspective for clinical therapy. Methods. Human chondrocyte cell line CHON-001 was administrated by different doses of interleukin (IL)-1β to simulate inflammation. Cell viability, migration, apoptosis, IL-6, IL-8, and tumour necrosis factor (TNF)-α expression, as well as expression of apoptosis-related factors, were measured to assess inflammation. miR-126 expression was measured by quantitative polymerase chain reaction (qPCR). Cells were then transfected with miR-126 inhibitor to assess the effect of miR-126 on IL-1β-injured CHON-001 cells. Expression of B-cell lymphoma 2 (Bcl-2) and the activity of mitogen-activated protein kinase (MAPK) / Jun N-terminal kinase (JNK) signaling pathway were measured by Western blot to explore the underlying mechanism through which miR-126 affects IL-1β-induced inflammation. Results. After IL-1β administration, cell viability and migration were suppressed while apoptosis was enhanced. Expression of IL-6, IL-8, and TNF-α were all increased, and miR-126 was upregulated. In IL-1β-administrated CHON-001 cells, miR-126 inhibitor suppressed the effect of IL-1β on cell viability, migration, apoptosis, and inflammatory response. Bcl-2 expression was negatively regulated with miR-126 in IL-1β-administrated cells, and thus affected expressions of phosphorylated MAPK and JNK. Conclusion. IL-1β-induced inflammatory markers and miR-126 was upregulated. Inhibition of miR-126 decreased IL-1β-induced inflammation and cell apoptosis, and upregulated Bcl-2 expression via inactivating the MAKP/JNK signalling pathway. Cite this article: C. D. Yu, W. H. Miao, Y. Y. Zhang, M. J. Zou, X. F. Yan. Inhibition of miR-126 protects chondrocytes from IL-1β induced inflammation via upregulation of Bcl-2. Bone Joint Res 2018;7:414–421. DOI: 10.1302/2046-3758.76.BJR-2017-0138.R1

As high incidences of tendinopathies are observed particularly in those who intensively use their tendons, we assume that pathological changes are caused, at least partially, by mechanical overload. This has led to the so-called overload hypothesis, explaining the development of tendinopathies by structural failure resulting from excessive load. At the same time, tendon loading is an important part in tendon rehabilitation. Currently, exercise treatment approaches such as eccentric training or heavy load resistance training are widely applied in tendinopathy rehabilitation, with good clinical results such as an improvement in function and a reduction in pain. Particularly those rehabilitative approaches which impose high strains on the tendon may induce an adaptation of the tendon's mechanical properties such as increased tendon stiffness. An increased tendon stiffness is often interpreted as desirable, as it may protect the tendon from overloading and thus prevent future strain injuries. However, the tendinopathic tendon is not necessarily less stiff than the tendon in the contralateral leg and an improvement in tendon stiffness is not necessarily accompanied by an improvement in tendon pain or function. In addition, metabolic factors, resulting e.g. in low-level systemic inflammation, may contribute to pathological tendon tissue changes and are not necessarily affected by an exercise program, while nutritional interventions or dietary supplements may potentially affect tendon cell metabolism. Indeed, dietary supplements have been introduced as an additional therapeutic approach in the treatment of tendinopathies in recent years, and their positive curative effects have been reported for both the general population and athletes. In the management of tendinopathies, it may thus be advisable if therapeutic approaches aim to address both tendon mechanics and tendon metabolism for better treatment effectiveness and a sustainable improvement in pain and function

In cartilage tissue engineering (TE),new solutions are needed to effectively drive chondrogenic differentiation of mesenchymal stromal cells in both normal and inflammatory milieu. Ultrasound waves represent an interesting tool to facilitate chondrogenesis. In particular, low intensity pulsed ultrasound (LIPUS)has been shown to regulate the differentiation of adipose mesenchymal stromal cells. Hydrogels are promising biomaterials capable of encapsulating MSCs by providing an instructive biomimetic environment, graphene oxide (GO) has emerged as a promising nanomaterial for cartilage TE due to its chondroinductive properties when embedded in polymeric formulations, and piezoelectric nanomaterials, such as barium titanate nanoparticles (BTNPs),can be exploited as nanoscale transducers capable of inducing cell growth/differentiation. The aim of this study was to investigate the effect of dose-controlled LIPUS in counteracting inflammation and positively committing chondrogenesis of ASCs embedded in a 3D piezoelectric hydrogel. ASCs at 2*10. 6. cells/mL were embedded in a 3D VitroGel RGD. ®. hydrogel without nanoparticles (Control) or doped with 25 µg/ml of GO nanoflakes and 50 µg/ml BTNPs.The hydrogels were exposed to basal or inflammatory milieu (+IL1β 10ng/ml)and then to LIPUS stimulation every 2 days for 10 days of culture. Hydrogels were chondrogenic differentiated and analyzed after 2,10 and 28 days. At each time point cell viability, cytotoxicity, gene expression and immunohistochemistry (COL2, aggrecan, SOX9, COL1)and inflammatory cytokines were evaluated. Ultrasound stimulation significantly induced chondrogenic differentiation of ASCs loaded into 3D piezoelectric hydrogels under basal conditions: COL2, aggrecan and SOX9 were significantly overexpressed, while the fibrotic marker COL1 decreased compared to control samples. LIPUS also has potent anti-inflammatory effects by reducing IL6 and IL8 and maintaining its ability to boost chondrogenesis. These results suggest that the combination of LIPUS and piezoelectric hydrogels promotes the differentiation of ASCs encapsulated in a 3D hydrogel by reducing the inflammatory milieu, thus representing a promising tool in the field of cartilage TE. Acknowledgements: This work received funding from the European Union's Horizon 2020 research and innovation program, grant agreement No 814413, project ADMAIORA (AdvanceD nanocomposite MAterIals for in situ treatment and ultRAsound-mediated management of osteoarthritis)

When joints sustain injury, the release of inflammation cytokines can cleavage matrix proteins and result in cartilage degradation and the subsequent osteoarthritis. RNA therapeutics emerging recently is a very promising approach to efficiently and specifically inhibit disease gene expression. However, the major challenge is how to deliver therapeutic RNA into joint and cartilage. Janus base nanotubes are self-assembled from synthetic Janus bases inspired from DNA base pairs. Based on the charge interaction, we are able to “sandwich” small RNAs among Janus base nanotubes to form tiny, nano-rod shaped delivery vehicles. Such vehicles can be engineered into different sizes and shapes. We have found that short and slim morphologies can greatly increase their penetration to extracellular matrix and delivery into “difficult-to-reach” tissues, such as cartilage and brain. Moreover, by delivering therapeutic siRNA, we have demonstrated its high-efficacy in inhibiting expression of an inflammatory regulator, Interleukin-1 receptor (IL-1R) in articular cartilage. Moreover, the inhibition effect is long-lasting so that joint inflammation and cartilage degradation caused by meniscus injury are greatly inhibited in a mouse model. Therefore, the Janus base nanotubes present a great potential in engineering into nano-structures for RNA delivery. Such approach may become an effective therapeutic against joint inflammation and arthritis

Abstract. OBJECTIVE. Changes in subchondral bone are one of few disease characteristics to correlate with pain in OA. 1. Profound neuroplasticity and nociceptor sprouting is displayed within osteoarthritic (OA) subchondral bone and is associated with pain and pathology. 2. The cause of these neural changes remains unestablished. Correct innervation patterns are indispensable for bone growth, homeostasis, and repair. Axon guidance signalling factor, Sema3A is essential for the correct innervation patterning of bony tissues. 3. , expressed in osteocytes. 4. and known to be downregulated in bone OA mechanical loading. 5. Bioinformatic analysis has also shown Sema3a as a differentially expressed pathway by bone in human OA patients. 6. HYPOTHESIS: Pathological mechanical load and inflammation of bone causes dysregulation of Sema3A signalling leading to perturbed sensory nerve plasticity and pain. METHODS. Human KOLF2-C1 iPSC derived nociceptors were generated by TALEN-mediated insertion of transcription factors NGN2+Brn3A and modified chambers differentiation protocol to produce nociceptor-like cells. Nociceptor phenotype was confirmed by immunocytochemistry. Human Y201-MSC cells were embedded in 3D type-I collagen gels (0.05 × 106 cell/gel), in 48-well plates and silicone plates, were differentiated to osteocytes for 7 days before stimulation with IL-6 (5ng/ml) and soluble IL-6 receptor (sIL-6r (40ng/ml), IL6/sIL6r and mechanical load mimetic Yoda1 (5μM) or unstimulated (n=5/group) (48-well plates) or were mechanically loaded in silicone plates (5000μstrain, 10Hz, 3000 cycles) or not loaded (n=5/group). Conditioned media transfer was performed from osteocyte to nociceptor cultures assessed by continuous 24-hour phase contrast confocal microscopy. 24-hours after stimulation RNA was quantified by RT-qPCR (IL6) or RNAseq whole transcriptome analysis/DEseq2 analysis (Load). Protein release was quantified by ELISA. Normally distributed data with homogenous variances was analysed by two-tailed t test. RESULTS. IPSC-derived nociceptor-like cells display elongated (>5mm) dendritic projections and nociceptive molecular markers such as TUJ1, PrPH and Neun and TrkA. Sema3A signalling ligands were expressed in 100% of osteocyte cultures. Mechanical loading regulated the Sema3 pathway; Sema3A (0.4-fold, p<0.001), Sema3B (13-fold, p<0.001), Sema3C (0.4-fold, p<0.001). Under inflammatory stimulation by IL6/IL6sR, SEMA3A (7-fold, p=0.01) and receptor Plexin1 (3-fold, p=0.03) show significant regulation. Sema3A protein release showed a significant downregulation of Sema3A release by IL6/sIL6r+Yoda1 (2-fold, p=0.02). Continuous 24-hour phase contrast confocal microscopy measuring the number of extending/retreating dendritic projections revealed that sensory nerve cultures exposed to media from osteocytes stimulated with IL-6/sIL-6R+Yoda1 displayed significantly more invading dendritic projections (p=0.0175, 12-fold±SEM 3.5) across 3 random fields of view within a single stimulated neural culture and significantly fewer retracting dendritic projections (p=0.0075, 2-fold±SEM 0.33) compared to controls. CONCLUSIONS. Here we show osteocytic regulation of Sema3A under pathological mechanical loading and the ability of media pathologically loaded osteocyte cultures to induce the branching and invasion of cultured nociceptor-like cells as displayed in OA subchondral bone. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Osteoarthritis (OA) is a common cause of chronic pain. Subchondral bone is highly innervated, and bone structural changes directly correlate with pain in OA. Mechanisms underlying skeletal–neural interactions are under-investigated. Bone derived axon guidance molecules are known to regulate bone remodelling. Such signals in the nervous system regulate neural plasticity, branching and neural inflammation. Perturbation of these signals during OA disease progression may disrupt sensory afferents activity, affecting tissue integrity, nociception, and proprioception. Osteocyte mechanical loading and IL-6 stimulation alters axon guidance signalling influencing innervation, proprioception, and nociception. Human Y201 MSC cells, embedded in 3D type I collagen gels (0.05 × 106 cell/gel) in 48 well plastic or silicone (load) plates, were differentiated to osteocytes for 7 days before stimulation with IL-6 (5ng/ml) with soluble IL-6 receptor (sIL-6r (40ng/ml) or unstimulated (n=5/group), or mechanically loaded (5000 μstrain, 10Hz, 3000 cycles) or not loaded (n=5/group). RNA extracted 1hr and 24hrs post load was quantified by RNAseq whole transcriptome analysis (NovaSeq S1 flow cell 2 × 100bp PE reads and differentially expressed neurotransmitters identified (>2-fold change in DEseq2 analysis on normalised count data with FDR p<0.05). After 24 hours, extracted IL-6 stimulated RNA was quantified by RT-qPCR for neurotrophic factors using 2–∆∆Ct method (efficiency=94-106%) normalised to reference gene GAPDH (stability = 1.12 REfinder). Normally distributed data with homogenous variances was analysed by two-tailed t test. All detected axonal guidance genes were regulated by mechanical load. Axonal guidance genes were both down-regulated (Netrin1 0.16-fold, p=0.001; Sema3A 0.4-fold, p<0.001; SEMA3C (0.4-fold, p<0.001), and up-regulated (SLIT2 2.3-fold, p<0.001; CXCL12 5-fold, p<0.001; SEMA3B 13-fold, p<0.001; SEMA4F 2-fold, p<0.001) by mechanical load. IL6 and IL6sR stimulation upregulated SEMA3A (7-fold, p=0.01), its receptor Plexin1 (3-fold, p=0.03). Neutrophins analysed in IL6 stimulated RNA did not show regulation. Here we show osteocytes regulate multiple factors which may influence innervation, nociception, and proprioception upon inflammatory or mechanical insult. Future studies will establish how these factors may combine and affect nerve activity during OA disease progression

Summary Statement. Antioxidant containing UHMWPE particles induced similar levels of in vitro macrophage proliferation and in vivo inflammation in the mouse air pouch model as UHMWPE particles alone. Benefit of antioxidant in reducing wear particle induced inflammation requires further investigation. Introduction. Wear particles derived from UHMWPE implants can provoke inflammatory reaction and cause osteolysis in the bone, leading to aseptic implant loosening. Antioxidants have been incorporated into UHMWPE implants to improve their long term oxidative stability. However it is unclear if the anti-inflammatory property of the antioxidant could reduce UHMWPE particle induced inflammation. This study evaluated the effect of cyanidin and vitamin E on UHMWPE induced macrophage activation and mouse air pouch inflammation. Methods. Four types of UHMWPE were used: (1) compression molded (CM) conventional GUR1020 (PE); (2) CM GUR1020 blended with 300 ppm cyanidin (C-PE); (3) CM GUR1020 blended with 1000 ppm α-tocopherol (BE-PE); and (4) CM GUR1020, gamma irradiated at 100kGy, diffused with α-tocopherol, and sterilised at 30kGy (DE-PE). Particles were generated by cryomilling. Particle count, size, and aspect ratio were determined using SEM and Image Pro. Each particle group was cultured with RAW264.7 macrophage cells at four concentrations (0.625, 1.25, 2.5, and 5 μg/mL) in a standard medium for 4 days. Cell numbers were quantified using MTT assay. Cytokine expression (IL-1β, TNFα, and IL-6) was measured using RT-PCR and ELISA. Particles were also suspended in PBS at 2 concentrations (0.2 or 1 mg) and injected into subcutaneous air pouches in BALB/c mice. Control animals were injected with PBS alone. Six days post-injection air pouches were harvested, half of which were fixed for histology to measure membrane thickness and inflammatory cell quantity. Remaining air pouches were frozen and analyzed by ELISA for cytokine production. Data were analyzed using one-way ANOVA with post hoc testing. P<0.05 was considered significant. Results. All 4 materials showed similar particle characteristics after cryomilling. Particle size ranged from 1 to 19 μm with 33% of particle population smaller than 2 μm. All particle groups supported macrophage proliferation, showing an inverse correlation between proliferation rate and particle dose. Gene expression of IL-1β and TNFα also showed an inverse correlation with particle dose. Expression of IL-1β, TNFα, and IL-6 appeared lower in cells cultured with C-PE than the other 3 materials. The accumulative protein productions of IL-1β and TNFα were significantly lower while IL-6 production was moderately lower in C-PE, BE-PE and DE-PE when compared to PE. Injection of polyethylene particles increased the air pouch membrane thickness significantly compared to the PBS control in all particle types and doses. Higher particle dose induced thicker membrane in all 4 materials. A similar trend was also observed in the percentage of inflammatory cell infiltration in the pouch membrane. C-PE and DE-PE particles at low dose and C-PE particles at high dose induced lower levels of IL-1β and TNFα than PE. IL-6 production was similar between PE and other 3 groups. Discussion/Conclusion. Antioxidant incorporated in UHMWPE did not alter the level of macrophage proliferation and air pouch inflammation induced by UHMWPE particles, although it reduced cytokine gene expression. Future investigation in a synovial joint environment is desired to evaluate the chronic inflammation response to antioxidant containing UHMWPE wear particles and to verify the effect of antioxidant in UHMWPE properties

Aim. This study aimed to assess whether the severity of symptoms (assessed with the Oxford Hip Score (OHS)) can relate to the levels of mRNA expression of markers for muscle inflammation (tumour necrosis factor alpha (TNFα), interleukin 6 (IL-6)) in the proximal vastus lateralis (VL) of patients with severe OA undergoing THR. Methods. Following local research ethics approval and informed consent, 17 patients were prospectively recruited. Muscle biopsies were obtained from the proximal VL (accessed through the surgical wound) intraoperatively whilst the OHS questionnaire was administered preoperatively. mRNA expression for TNFα and IL-6 was assessed using the reverse transcriptase polymerase chain reaction (RT-PCR). The median OHS was used for stratification, with patients above the median classed as having moderate symptoms (MS) and those below classed as having severe symptoms (SS). The effect of SS on muscle inflammation was assessed with relative quotient (RQ) comparison of SS vs. MS mRNA expression. Results. Patients recruited were (mean (SD)) 65.3(8.8) years old in men (n=9) and 59.8 (13.3) years old in women (n=9). The median OHS was 24 (range 10–32) with SS < 24 (n=10) and MS ≥ 24 (n=7). In comparison to the MS group, the SS group had increased TNFα expression (+28% (RQ=1.28, p=0.35)) with reduced IL-6 expression (−44% (RQ=0.56, p=0.35), though neither of these reached statistical significance. Conclusions. Muscle inflammation is not clearly correlated to symptoms in this group. Preoperative subjective functional deficit appears independent of muscle inflammation in patients with hip osteoarthritis

Summary Statement. We observed that severe muscle weakness leads to OA, whereas a transient inflammatory stimulus did not have a significant effect on cartilage degradation. This arises the thought that a severe but transient inflammation may not be an independent risk factor for OA. Introduction. Biomechanical disturbances and joint inflammation are known risk factors, which may provoke or advance osteoarthritis (OA). However, the effect of interactions of such risk factors on the onset and progression of OA are still poorly understood. Therefore, the goal of this study was to investigate the in vivo effects of muscle weakness, joint inflammation, and the combination of these two risk factors, on the onset and progression of OA in the rabbit knee. Patients & Methods. Thirty 1-year-old skeletally mature female New Zealand White rabbits (weight: average 5.7kg, range 4.8–6.6kg) were used in this study. The animals were divided into four experimental groups: (i) surgical transection of the nerve branch of the common femoral nerve leading to the vastus lateralis muscle; (ii) muscle weakness of the quadriceps muscle induced by a chronic intramuscular injection of Botulinum toxin A (BTX-A) (3); (iii) intraarticular injection in the experimental knee joint with commercially available sterile Carrageenan solution to induce a transient severe inflammatory reaction (4); (iv) administration of both intraarticular injection of Carrageenan and intramuscular injection of BTX-A. In each animal, one hind limb was randomly assigned to the experimental intervention, while the contralateral side acted as its own control. Ninety days following intervention, muscle mass, joint diameter and cartilage histology of the femur, femoral groove, tibia and patella were assessed and microscopically analyzed using the OARSI histology score. Results. Transection of the femoral branch leading to the vastus lateralis as well as the administration of BTX-A led to a significant muscle mass loss for the vastus lateralis and the total quadriceps group, respectively. Similar results were seen in the combined Carrageenan/BTX-A group. There were no changes in total quadriceps muscle mass in the Carrageenan group. Knee joint diameters of the experimental limb were significantly increased in the Carrageenan and Carrageenan/BTX groups. VL transection and BTX-A injection did not cause significant increases in joint diameter. Histologic assessment of the cartilage showed that weakness of the vastus lateralis resulted in significantly higher OARSI scores in the patella and femoral groove, but not the tibiofemoral articulation. The administration of BTX-A caused significant cartilage damage in all 4 compartments (patella, femur, tibia, femoral groove). Intraarticular injection of Carrageenan did not cause significant cartilage damage in any compartment compared to the contralateral side. The combination of BTX-A and Carrageenan resulted in severe cartilage damage in the patella in all four compartments of the knee. The most severe damage was found on the medial side of the tibiofemoral joint and the lateral side of the patellofemoral joint. Conclusion. Severe muscle weakness over a three months period leads to the onset and progression of OA in the rabbit knee. A transient local inflammatory stimulus did not promote cartilage degradation, nor did it enhance cartilage degradation when it was combined with muscle weakness. This result is surprising and adds to the literature the idea that a severe but transient inflammation may not be an independent risk factor for OA

Osteoarthritis (OA), characterised by pain, disability and joint degeneration, is common and has no cure. Prevalence of severe radiographic knee OA is 19% in over 45's and 50% in over 75's in the US and Europe. Abnormal joint loading, or injury, increase risk of OA. We have discovered that glutamatergic signalling is mechanically regulated and glutamate receptors (GluR) drive inflammation, degeneration and pain representing potential drug targets in osteoarthritic joints. Joints from OA and knee injured patients, and rodent models of arthritis, show increased synovial fluid glutamate concentrations and abundant GluR expression. Since AMPA/kainate GluRs regulate IL-6, a critical mediator of arthritic degeneration, we tested protective effects of the AMPA/KA GluR antagonist, NBQX in animal models of arthritis. In rodent antigen induced arthritis, and osteoarthritis (meniscal transection and anterior cruciate ligament rupture), NBQX reduced joint swelling, degeneration and pain, exceeding anti-degenerative effects of other drugs tested similarly. 3D osteocyte/osteoblast co-cultures and human bone samples taken from patients undergoing high tibial osteotomy joint realignment surgery, revealed underlying cellular mechanisms mediated by bone cells. Related drugs, already used in humans for epilepsy and migraine, represent a repurposing opportunity and are effective in our models of arthritis

The purpose of this study was to evaluate the beneficial effects of r-Irisin (IR) on human primary tenocytes (hTCs) in vitro. Indeed, Irisin is secreted from muscles in response to exercise and mediates many beneficial effects on tissues and organs.

Tissue samples (n=3) were analyzed by histology and immunohistochemistry for αVβ5 receptor. hTCs isolated, culture expanded were treated with: 1) RPMI medium as control; 2) IR at different concentrations; 3) IL-1β; 4) pre-treated with IL-1β for 24 h and then co-treated with IR; 5) pre-treated with IR for 24 h and then co-treated with IL-1β. We evaluated: cell metabolic activity (MTT); cell proliferation (trypan blue staining and PicoGreen); nitrite concentration (Griess). The analysis were performed in triplicate for each donor and each experiment was repeated at least three times. Data were expressed as mean ± S.D. One-way ANOVA analysis was used to compare the groups under exam.

We found the presence of the αVβ5 receptor on hTCs plasma membrane supporting the potential interaction with irisin. Cell proliferation was significantly increased with IR at 5, 10 and 25 ng/mL. IR 25 ng/mL after IL1β pre-treatment was able to counteract the increase of nitrite production (p < 0.001) compared to the inflamed hTCs (p < 0.01; p < 0.0001), as well as IR at 10 and 25 ng/ml showed a protective role from oxidative damage. We observed a significant increase in cell metabolic viability in culture under IR at 5 and 25 ng/mL (p < 0.001; p < 0.05) in the pre-treated IR groups, whereas IR showed anti-inflammatory effects at the highest concentration of r-Irisin (p < 0.05).

This is the first study reporting the capability of irisin to attenuate tendinopathy in vitro by acting on acute inflamed tenocytes. Our results confirmed and highlighted the potential cross-talk mechanism between muscle and tendon.